Apolipoprotein E (ApoE) is a 34,000 molecular weight protein which is the product of a single gene on chromosome 19. Human ApoE exists in three major isoforms designated apoE2, apoE3 and apoE4 (for review, see Mahley (in press) Molecular and Genetic Bases of Neurological Disease 2nd ed.; and Mahley Science 240:622-630 (1988)). The different isoforms result from amino acid substitutions at amino acid residue positions 112 and 158. The common isoform, apoE3, has a cysteine residue at position 112 and an arginine residue at position 158. The apoE4 isoform differs from apoE3 only at position 112, which is an arginine residue. The apoE2 isoform, associated with type III hyperlipoproteinemia (Mahley (1988)), differs from apoE3 only at position 158, which is a cysteine residue. ApoE3 and apoE4 bind normally to the low density lipoprotein (LDL) receptor, whereas apoE2 does not.
ApoE contains two structural domains, an amino-terminal and a carboxy-terminal domain, each associated with a specific function. Weisgraber Adv. Protein Chem. 45:249-302 (1994). The amino terminal domain contains the lipoprotein receptor binding region and the carboxy-terminal domain contains the major lipid-binding elements. The two domains appear to interact with each other in an isoform-specific manner such that amino acid substitutions in one domain influence the function of the other domain, a phenomenon referred to as domain interaction. Domain interaction is responsible for the preference of apoE4 for very low density lipoproteins (VLDL) contrasted with the preference of apoE3 for high density lipoproteins (HDL). The specific amino acid residues in apoE4 that are involved in this interaction are arginine-61 in the amino-terminal domain and glutamic acid-255 in the carboxy-terminal domain. Dong et al. J. Biol. Chem. 269:22358-22365 (1994); and Dong and Weisgraber Circulation 92:I-427-I-428 (1995)(abstract).
By redistributing lipids among the cells of different organs, apoE plays a critical role in lipid metabolism. While apoE exerts this global transport mechanism in chylomicron and VLDL metabolism, it also functions in the local transport of lipids among cells within a tissue. Cells with excess cholesterol and other lipids may release these substances to apoE-lipid complexes or to HDL containing apoE, which can transport the lipids to cells requiring them for proliferation or repair. The apoE on these lipoprotein particles mediates their interaction and uptake via the LDL receptor or the LRP.
ApoE also functions in a neurobiological role. ApoE mRNA is abundant in the brain, where it is synthesized and secreted primarily by astrocytes. Elshourbagy et al. Proc. Natl. Acad. Sci. USA 82:203-207 (1985); Boyles et al. J. Clin. Invest. 76:1501-1513 (1985); and Pitas et al. Biochem. Biophys. Acta 917:148-161 (1987). The brain is second only to the liver in the level of apoE mRNA expression. ApoE-containing lipoproteins are found in the cerebrospinal fluid and appear to play a major role in lipid transport in the central nervous system (CNS). Pitas et al. J. Biol. Chem. 262:14352-14360 (1987). In fact, the major cerebrospinal fluid lipoprotein is an apoE-containing HDL. ApoE promotes marked neurite extension in dorsal root ganglion cells in culture. Handelmann et al. J. Lipid Res. 33:1677-1688 (1992). ApoE levels dramatically increase (about 250-fold) after peripheral nerve injury. Meller et al. Science 228:499-501 (1985); and Ignatius et al. Proc. Natl. Acad. Sci. USA 83:1125-1129 (1986). ApoE appears to participate both in the scavenging of lipids generated after axon degeneration and in the redistribution of these lipids to sprouting neurites for axon regeneration and later to Schwann cells for remyelination of the new axons. Boyles et al. J. Clin. Invest. 83:1015-1031 (1989); and Ignatius et al. Science 236:959-962 (1987).
More recently, apoE has been implicated in Alzheimer's disease (hereafter "AD") and cognitive performance. Saunders et al. Neurol. 43:1467-1472 (1993); Corder et al. Science 261:921-923 (1993); and Reed et al. Arch. Neurol. 51:1189-1192 (1994). ApoE4 is associated with the two characteristic neuropathologic lesions of AD: extracellular neuritic plaques representing deposits of amyloid beta (AJ) peptide; and intracellular neurofibrillary tangles representing filaments of hyperphosphorylated tau, a microtubule-associated protein. For review, see, McKhann et al. Neurol. 34:939-944 (1984); Selkoe Neuron 6:487-498 (1991); Crowther Curr. Opin. Struct. Biol. 3:202-206 (1993); Roses Curr. Neurol. 14:111-141 (1994); Weisgraber et al. Curr. Opin. Lipidol. 5:110-116 (1994); and Weisgraber et al. Curr. Opin. Struct. Biol. 4:507-515 (1994).
AD is generally divided into three categories: early-onset familial disease (occurring before 60 years of age and linked to genes on chromosomes 21 and 14); late-onset familial disease; and sporadic late-onset disease. Both types of late-onset disease have recently been linked to chromosome 19 at the apoE locus. Other results suggest that apoE4 is directly linked to the severity of the disease in late-onset families. Roses (1994). Recently, cholesterol lowering drugs, the statins, have been suggested for use in treating AD by lowering apoE4 levels. WO 95/06470.
The neurofibrillary tangles, which are paired helical filaments of hyperphosphorylated tau, accumulate in the cytoplasm of neurons. Tau is a microtubule-associated phosphoprotein which normally participates in microtubule assembly and stabilization; however, hyperphosphorylation impairs its ability to interact with microtubules. Increased binding of tau by apoE has been suggested as a treatment for AD. WO 95/06456.
In vitro tau interacts with apoE3, but not with apoE4. Strittmatter et al. Exp. Neurol. 125:163-171 (1994). The interaction of apoE3 with tau may prevent its hyperphosphorylation, thus allowing it to function normally in stabilizing microtubular structure and function. In the presence of apoE4, tau could become hyperphosphorylated and thus inactive, which could promote the formation of neurofibrillary tangles.
ApoE4 has recently been associated with decreased learning ability and impaired memory. Helkala et al. Neurosci. Letts. 191:141-144 (1995). ApoE4 has been found to be a strong predictor of the outcome of patients designated as having memory impairment. Note that, apoE4 has been described as a risk factory, rather than a diagnostic. Peterson et al. JAMA 273:1274-1278 (1995); and Feskens et al. BMJ 309:1202-1206 (1994).
ApoE3 and apoE4 are also though to play a role in neurite repair and remodeling in the CNS. In cultured neurons, apoE3 stimulates neurite extension, whereas apoE4 inhibits neurite extension. Nathan et al., 1994. Repair and remodeling of neurons in response to stress or injury, either chronic or acute, should thus proceed more effectively in the presence of apoE3 than in the presence of apoE4.
There are currently no effective therapies for arresting (and, more importantly, reversing) the impairment of central and peripheral nervous system function once a degenerative cascade begins. Likewise, there is no current therapy for restoration of normal, central and peripheral nervous system function when the induced stress has a less catastrophic or partially reversible effect compared to the dementias. The effects of events that impair the function of the CNS, such as traumatic brain injury and stroke are in need of such therapies to mitigate or reverse the resulting damage. In addition, no effective therapies for reducing or reversing impairment in cognitive learning and behavior are known, as relatively little is understood about the mechanisms of cognitive learning and memory.